Lipase activity in serum or plasma is increased in pancreatic diseases (e.g., acute and chronic pancreatitis or pancreatic cancer), so that the lipase activity is a useful marker for the pancreatitis, etc.
The lipase is an enzyme that catalyzes a reaction in which ester bonds (at α-position (positions 1 and 3)) of a triglyceride (TG) (three long-chain fatty acid molecules are each linked via an ester bond to glycerol) are hydrolyzed to produce two fatty acid molecules and one β-monoglyceride molecule.
This one β-monoglyceride molecule is isomerized to α-monoglyceride, which is then hydrolyzed by the lipase to produce a glycerol and a fatty acid.
Examples of an assay for measuring lipase activity in serum or plasma include the following assays (see Non Patent Documents 1 and 2).
The examples known include a Cherry-Crandall method in which an olive oil emulsion is used as a lipase substrate; this olive oil emulsion is made to contact and react with, for example, a serum sample at 37° C. for 24 hours; and then a fatty acid, which has been generated through hydrolysis by the lipase, is titrated with an alkali.
In this method, however, the reaction time is long, the lipase of interest can be inactivated, and the reaction is thus markedly inhibited.
Another example known is a Vogel-Zieve method and a modified method thereof in which a triolein or olive oil emulsion is used as a lipase substrate; this triolein or olive oil emulsion is made to contact and react with, for example, a serum sample; the emulsified micelles are then hydrolyzed by the lipase to cause a decrease in turbidity of the resulting reaction solution; and the lipase activity is determined from the decrease.
These methods, however, involve serum protein-mediated inhibition and/or interference due to rheumatoid factor-induced aggregation, so that it is difficult to produce a uniform and stable emulsion. Also, the methods are poorly reproducible and thus disadvantageous.
Another example known is an assay for measuring lipase activity in which BALB (2,3-dimercapto-1-propanol tributyrate) is used as a lipase substrate; this BALB is made to contact and react with, for example, a serum sample; BAL (2,3-dimercapto-1-propanol), which has been generated through hydrolysis by the lipase, is reacted with DTNB (5,5′-dithiobis-2-nitrobenzoic acid); and yellow light emitted by the resulting TNB anion is measured at 412 nm.
This assay, however, involves interference with a liver esterase under highly concentrated conditions. Accordingly, the liver esterase is mixed through a reaction cell or a nozzle (probe) from an assay reagent for measuring another item. This affects a measured value and causes an error, so that this assay is thus disadvantageous.
Another example known is an assay for measuring lipase activity in which 1,2-dilinoleoyl glycerol, which is a natural substrate, is used as a lipase substrate; this 1,2-dilinoleoyl glycerol is made to contact and react with, for example, a serum sample: linoleic acid, which has been generated through hydrolysis by the lipase, cooperates, in the presence of Coenzyme A, NAD+, and ATP, with acyl-CoA synthetase, Acyl-CoA oxidase, and an enoyl-CoA hydratase-3-hydroxyacyl-CoA dehydrogenase-3-ketoacyl-CoA thiolase multienzyme complex to perform β-oxidation; and then the NADH production rate when the β-oxidation occurs is measured.
This assay, however, also involves interference with a liver esterase under highly concentrated conditions. Accordingly, the liver esterase is mixed through a reaction cell or a nozzle (probe) from an assay reagent for measuring another item. This affects a measured value and causes an error, so that this assay is thus disadvantageous.
In addition to the above respective assays, an assay for measuring lipase activity in serum or plasma has been developed in which 1,2-o-dilauryl-rac-glycero-3-glutaric acid (6′-methylresorufin) ester (DGGMR) is used as a lipase substrate (see Patent Document 1 and Non Patent Document 2).
In this assay, 1,2-o-dilauryl-rac-glycero-3-glutaric acid (6′-methylresorufin) ester (hereinafter, sometimes referred to as “DGGMR”) is made to contact and react with, for example, a serum sample; and the lipase catalyzes hydrolysis to generate 1,2-o-dilauryl-rac-glycerol and glutaric acid (6′-methylresorufin) ester.
This glutaric acid (6′-methylresorufin) ester is unstable and is hydrolyzed readily and naturally to generate 6′-methylresorufin (λmax: 580 nm).
An increase in the 6′-methylresorufin generated is measured by reading absorbance at or near 580 nm. By doing so, the lipase activity value in the sample can be determined.
This assay for measuring lipase activity using DGGMR as a lipase substrate is simple because the measurement proceeds in a series of reactions. Besides, the assay is also advantageous because the assay is unlikely to be affected by an esterase mixed through a reaction cell or a nozzle (probe) from other measuring reagents.
Meanwhile, a lipase contained in, for example, serum or plasma is most efficient at a water-oil interface of an emulsified triglyceride substrate. The reaction rate of this lipase involves the surface area of the substrate dispersed. Thus, for measuring the lipase activity, it seems critical to prepare a substrate composed of stable and uniform micelle particles (see Non Patent Document 2).
For this purpose, when a substrate solution (substrate solution for measuring lipase activity) used for measuring lipase activity is produced conventionally, the substrate solution should be emulsified and composed of stable and uniform micelle particles. To realize this, various methods have been taken into consideration: a substrate may be mixed into an aqueous solution containing a surfactant; a substrate may be mixed into a solution containing an organic solvent (e.g., an alcohol); a substrate-containing liquid may be added dropwise and mixed into a solution; a substrate-containing liquid may be jet-injected into a solution; a substrate solution may be stirred with a powerful mixer at a high speed; or a substrate solution may be subject to ultrasonication. The methods necessitate cumbersome or special processing such that skill is required. The methods also necessitate a special apparatus, instruments, or other items.
For example, disclosed is a process for producing a transparent miscible aqueous solution containing a water-insoluble substance, characterized in that a water-insoluble substance (e.g., triglyceride) as a lipase substrate is added to an aqueous solution containing a nonionic surfactant; the mixture is heated while being stirred; the temperature is once raised to a temperature higher than the clouding point of the nonionic surfactant; and the temperature is then cooled to a temperature equal to or less than the clouding point while the mixture is further stirred (see Patent Document 2).
Also disclosed is a transparent triglyceride substrate solution for measuring lipase activity, characterized in that an aqueous solution containing a nonionic surfactant is heated to a temperature equal to or higher than the clouding point of the nonionic surfactant; a triglyceride is added and dissolved in the mixture while being stirred to prepare a uniform and miscible (transparent) aqueous solution containing the triglyceride; and the resulting aqueous solution is used as a lipase substrate and, as needed, further includes a lipase function promoter (see Patent Document 3).
Also disclosed is a process for producing a substrate solution used for measuring lipase activity in a sample, characterized in that when a substrate (e.g., a triglyceride) is mixed with a surfactant (e.g., a nonionic surfactant with an HLB of 10 to 16), an water-soluble organic solvent (e.g., methanol or ethanol) is mixed together; and a mixing process uses vibrations such as ultrasonication, wherein the substrate solution contains micelles with a geometric mean diameter of 0.17 μm to 0.38 μm and the geometric standard deviation of the diameter distribution of the micelles is 0.25 μm or less (see Patent Document 4).
Here, in the assay for measuring lipase activity using the DGGMR as a lipase substrate, the substrate solution is produced as follows: “b) 0.9 g of sodium taurodeoxycholate and 0.3 g of a colipase (from a pig) are dissolved under stirring in 60 ml of distilled water. While being well stirred, a solution containing 70 mg of 1,2-o-dilauryl-rac-glycero-3-glutaric acid (6-methyl-resorufin) ester in 1.7 ml of n-propanol is jet-injected into the above solution” (Example 29 of Patent Document 1); or “Reagent 2: 0.6 g of a chromogenic substrate for a lipase (e.g., 1,2-o-dilauryl-rac-glycero-3-glutaric acid (6′-methylresorufin) ester) was dissolved into 9 ml of a suitable alcohol (e.g., ethanol). Then, 1 g of an emulgator (e.g., Brij 35 or Triton X-114) was added to the solution. The resulting oelic phase was aspirated using an injection needle and was made to pass through a fine cannula (with an inner diameter of 0.15 to 1.0 mm) under a high pressure, so that the solution is pressure-injected into an aqueous solution under stirring” (Example 3 of Patent Document 5). In view of the above, an alcohol was used as an organic solvent and the assays necessitate a process in which the substrate-containing liquid is jet-injected (injected under a high pressure) into a solution.
Note that disclosed is that a substrate solution for lipase analysis contains a lipase substrate (e.g., DGGMR) as well as at least one lipase substrate solubilizer selected from an anionic surfactant, a lecithin, and a cholesterol ester. This substrate solution exerts an effect of making the substrate well miscible without decreasing the activity of the substrate (see Patent Document 6).
Then, this document describes results where when 0.1% by weight of a nonionic surfactant was used instead of the above lipase substrate solubilizer, “the solution was turbid and thus had a drawback in the measurement”.
Also disclosed is a lipase substrate solution for measuring enzyme activity, characterized by containing at least a lipase substrate (e.g., DGGMR) and 1,2-diphytanoyl-sn-glycero-3-phosphocholine as a lipase substrate solubilizer. This substrate solution is very transparent. Accordingly, effects can be exerted such that the lipase activity can be measured with high accuracy and its storage stability is increased (see Patent Document 7).